This is an ongoing comprehensive program for study at the molecular level of composition and mechanistic details of mitochondrial electron transport; energy conservation, transduction and transfer; ATP synthesis and hydrolysis. Five enzyme complexes have been isolated: I, II, III and IV are segments of the respiratory chain and capable of total respiratory activity reconstitution; V catalyzes oligomycin- and uncoupler-sensitive ATP-Pi exchange at 350-470 nmoles min-1.mg-1 protein, and contains the mitochondrial uncoupler-binding protein as determined by equilibrium binding and photoaffinity labeling studies, using radioactive 2-azido-4-nitrophenol. The purpose of the proposed research is in complex I: to study mechanisms of NADH and NADPH oxidation and ubiquinone (Q) reduction, isolate and purify the iron-sulfur proteins bearing centers 1, 2, 3, 4, study mechanisms of energy conservation and transfer; in complex II: to study mechanism of Q reduction, roles of cytochrome b557.5 and the low-potential iron-sulfur center S-2, purify b557.5; in complex III: to purify cytochromes b562 and b566, study their regulatory properties, study and better characterize chromophore-558, study mechanism of energy conservation and transfer; in complex V: to study mechanisms of ATP-Pi exchange, ATP hydrolysis, uncoupling, energy conservation and transfer, components required for these functions and the role of each in ATP synthesis and hydrolysis, the role of the ATPase protein in regulation of ATPase function, reconstitution of highly purified V with I, III and IV for energy transfer and oxidative phosphorylation. This supplemental application requests funds for an air compressor, a cold room, a fume hood, and minor alterations, all necessitated by our move in July, 1980, from the present location to the new building of the institution.